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ACADEMY OF VETERINARY, VETERINARY FOOD SAFETY PROGRAMME, DEPARTMENT OF ANATOMY AND PHYSIOLOGY, DEPARTMENT OF ANATOMY AND PHYSIOLOGY, RAIMONDA AURYLĖ, Investigation of the FERMENTATION of MILK OF DIFFERENT LACTOBACIAL SPECIES, MAISTER's thesis, Supervisor: Prof. Dr. Vaidas Oberauskas

 1.3. Probiotics

Probiotics are live micro-organisms, microbial food supplements with a positive and natural effect on the microflora of the gastrointestinal tract (Abe et al. 1995; Fuller, 1989). 

The term "probiotics" was first used in 1965 by Lilly and Stillwel to describe the effect of one micro-organism acting against another. The term has since been applied to live microbial supplements that improve the composition of the gastrointestinal microflora (Kačerauskis et al., 2003). Probiotics literally means 'for life'. This term now refers to living micro-organisms or products fermented with them that provide benefits to the human or animal body by maintaining or improving the balance of the intestinal microflora. Probiotic bacteria are generally defined by most experts as eubiotics (representatives of the normal microflora of the intestine and other body cavities, usually bifidobacteria and Lactobacillus genus lactic acid microflora), known as classical probiotics (Kačerauskis et al., 2003).

The most common strains of probiotics are: Lactobacillus - Lactobacillus acidophilus, L. casei, L. rhamnosus and Bifidobacterium, Enterococcus, Pediococcus, and Streptococcus strains of bacteria in the genera. Lactobacilluss are naturally found in milk, cereals and other plants (Quillien, 2001; Michalík et al., 1999; Fuller, 1989).

Around 400-500 genera of gut bacteria have been identified. The vast majority of gut bacteria are beneficial. One gram of large intestinal contents is home to 10 - 

1000 billion bacteria. In a complex interplay of different species, each type of bacteria plays a role, and the ratio of bacteria to each other affects health. Depending on which species dominate, our health depends on which species dominate. In the large intestine, the following genera predominate: Bacteroides, Bifidobacterium, Eubacterium and Peptostreptococcus. Smaller but equally important are Streptococcus, Lactobacillus, Enterococcus and Clostridium tribes and Enterobacteriaceae family. The gut contains bacteria that become harmful in large quantities, i.e. Veillonellae, Clostridium, Staphylococcus, Proteus and Pseudomonas. They lead to the formation of metabolic end-products such as ammonium, hydrogen sulphide, amines, phenols, indoles and secondary bile acids. These substances have an adverse effect when absorbed into the bloodstream and directly into the intestinal mucosa. Beneficial bacteria are much more abundant than harmful ones. The balance of the microbiota can be disturbed by the use of antibiotics, changes in dietary habits or gastrointestinal diseases. (www.valio.lt. Accessed 10.12.2012) 

These micro-organisms are added as food supplements to yoghurts, cottage cheese, kefir, or in the form of lyophilised powders (capsules, microcapsules), tablets and liquid suspensions (Reid et al., 2003). 

1.3.1. Properties of probiotics

There are billions of bacteria in the human body and they make up the human microbiota. Good bacteria are important for digestion and immunity. They are also important for the prevention and treatment of various diseases (Isolauri  et. al., 2002; Levy, 2000).  

Probiotics must have the following properties:

• Able to colonise specific body cavities by attaching to receptors on mucosal epithelial cells of the intestinal mucosa or other cavities;
• Be non-pathogenic, genetically stable, non-toxic;
• Produce antimicrobial agents and be antagonists towards other micro-organisms;
• Be resistant to acids and bile acids and to digestive enzymes, so that they can pass through the right parts of the digestive tract;
• Able to multiply in the body and restore the normal microbiota;
• Be able to activate the body's immune system;
• Eliminate pathogens or reduce the chances of their establishment; 
• To be able to maintain viability and stability during technological processes in the production of their particular formulations and to remain stable for a given period of time in the formulations produced;
• Meet the necessary requirements for normal food production, e.g. organoleptic properties, stability, viability, and not necessarily be involved in the fermentation of the product (Toit et al., 1998; Maragkoudakis et al., 2006; Masteikienė, 2006);
• Produce organic acids, hydrogen peroxide and bacteriocins that antagonise pathogen growth (Reid et al.,2003; Kaur et. al., 2002; Ouwehand et.al., 2004);
• Probiotics can also stimulate and accelerate metabolic processes in the gastrointestinal tract, which have a positive effect on the health of the body (Conway, 2001).

The most research evidence is on the benefits of probiotics in the treatment of intestinal infections. Probiotics have been shown to shorten the duration of diarrhoea. Probiotic bacteria may also help protect against diarrhoea or other acute gastrointestinal disorders (Sullivan, Nord, 2002). 

There are publications on the benefits of probiotics Helicobacter polora for the treatment and prevention of infections. H. pylori causes type B gastritis and peptic ulcers and is a risk factor for gastric cancer. Studies have shown that lactobacilli inhibit H. pylori growth and the urease activity it needs to survive in the acidic gastric environment. Other studies show that probiotics inhibit H. pylori infection and reduces the risk of recurrence. Experiments in mice have shown that Lactobacillus salivarius protects the stomach against H. pylori infection and inhibit it. Studies have shown that probiotics are highly effective in lactose intolerance (Pavilionis et al., 2005).

1.4. Lactobacillus

Lactobacillus are Gram-positive, rod-shaped, non-spore-producing and catalase-negative probiotic bacteria. They are one of the most important lactic acid bacteria used in food production and as probiotics (Ortua et. al. 2007).

The modern taxonomy of lactic acid probiotic bacteria includes lactobacilli, bifidobacteria and enterococci. Probiotic bacteria include 176 strains. They have certain requirements: the bacteria must be able to attach to the intestinal wall, be genetically stable, resistant to the environment, and reproduce well in vitro and in vivo (Klein et. al., 1998).

Over the last decade, the market for functional products containing probiotics has been the fastest growing area of new food product development. Functional foods require new probiotic cultures with beneficial physiological properties (Sieladie et. al.., 2011).

Milk and its products are one of the growth media for lactobacilli. The strain of lactic acid bacteria is determined by identification of morphological and biochemical indicators, such as Gram staining and catalase test (Sujaya, 2008). Survival at different temperatures, pH and salty environments is important for lactobacilli. 

The best studied micro-organisms at present are lactic acid-producing bacteria, especially Lactobacillus spp. and Bifidobacterium spp. (Rolfe, 2000). 

Lactobacilli are one of the largest strains of the lactic acid bacteria group, so named because they convert lactose and other sugars into lactic acid and are highly resistant to the gastrointestinal environment of stomach and bile acids (Rayid et. al., 2007; Khali et. al.., 2007; Abdelbasset et. al., 2008).

Lactobacillus rod-shaped bacteria, ranging in size from 1.0 to 10 x 0.5 to 1.2 μm, and ranging in shape from a stretched rod to a coccobacillus. These bacteria form short chains, especially in the later logarithmic phase of growth. Most lactobacilli are immobile, non-spore forming, Gram +, old cultures become Gram - as the pH of the medium increases. They are facultative anaerobes (some microaerophiles), negative for catalase and cytochrome oxidase. Faster growth of lactobacilli is observed in environments with 5 -10% CO₂ concentration. It grows better in the presence of certain substances - amino acids, peptides, fatty acids, vitamins, carbohydrates and nucleic acids. The optimum pH concentration is 5,5 - 5,8 and the growth temperature is 30 - 40 ⁰C, but grows at temperatures between +5 and +53 ⁰C (Axelsson, 1998). 

Probiotic lactic acid bacteria are found in a variety of ecological niches, such as plants, the digestive tract of animals and humans, the oral cavity, the vagina and raw milk. They are also found in water, sewage, soil, manure and silage (Hammes, Vogel, 1995). The most common foods are milk, cereal and meat products, vegetables, fruit, juice, wine, beer (Jaymanne et. al., 2006).

The genus Lactobacillus includes 133 species and 18 subspecies (Liu, 2011). Lactobacilli are divided into strict homofermentative (L. planarum, L.

acidophilus, L. casei. L. salivarium), strict heterofermentation and optional heterofermentation.

Homofermentative lactobacilli break down glucose, fructose, galactose, maltose, sorbitol and produce about 85% lactic acid during fermentation.

Heterofertile lactobacilli break down glucose, xylose, arabinose and rhamnose, resulting in a smaller amount of lactic acid (50%).

Facultative heterofermentative lactobacilli occupy an intermediate position. They cleave pentoses (arabinose, xylose, etc.) and hexoses (glucose, fructose, maltose, etc.) to form lactic acid, and when glucose is depleted, they break it down to acetic acid and alcohol (Wood and Holzapfel, 1998).

Lactobacilli are industrially used in the production of yoghurt, wine, cider, sauerkraut, pickles, cheese, chocolate and other fermented foods (Bixquert, 2009).

1.4.1. Lactobacillus acidophilus

Lactobacillus acidophilus are Gram-positive, spore-forming, homofermentative, catalase-negative rods. They grow in a medium with a relatively low pH (less than pH 5,0) and an optimum growth temperature of 37 ⁰C. (Bâati et. al. 2000). These bacteria live naturally in the human gastrointestinal tract, mouth and vagina, are also found in some fermented foods (e.g. kefir, yoghurt, etc.), and are used in the production of probiotic foods and food supplements.

Like most lactobacilli, L. acidophilus Ferments lactose into lactic acid. L. acidophilus the gut and vagina provide unfavourable conditions for the growth of pathogenic and relatively pathogenic bacteria. These probiotic bacteria are important for digestion, separating amino acids from the bile, thus facilitating the reuse of amino acids by the body. Scientific studies have shown that L. acidophilus lowers blood cholesterol, boosts natural immunity, relieves diarrhoea, increases resistance to fungal infections. Probiotic-containing L. acidophilus It is useful for balancing the intestinal microflora in a number of gastrointestinal disorders, such as diarrhoea, constipation, rotavirus infection, gastric or duodenal ulcers, ulcerative colitis, Crohn's disease, irritable bowel syndrome, candidiasis. 

Very rarely, these bacteria are associated with infections (Anderson, Gilliland 1999; Aguirre, Collins, 1993; Gasser, 1994; Salminen et. al. 1998; Borriello et. al. 2003).

In vitro studies have shown that L. acidophilus is highly resistant to low pH and can survive in the duodenum in the presence of bile acids. Interaction with the intestinal mucosa is important as binding to the intestinal mucosa allows probiotics to survive longer in the intestine. It can also protect against intestinal pathogens. It has been shown that L. acidophilus attaches very well to the epithelium of the human digestive tract.

More than 90 % of the EU's L. acidophilus remain viable for about 1 hour in a solution of hydrochloric acid and pepsin (1 %) at 37 ⁰C and pH 3.

More than 90 % of the EU's L. acidophilus survives in a medium containing 0.3% bile acid.

More than 60 % of the EU's citizens are. L. acidophilus survives for 1 hour in a medium containing 0,3 % pepsin at pH 2.

More than 60 percent of lactobacilli remain viable in a medium containing 0.1 percent pancreatin at a pH of 8 for 2 hours. (http://ss1.spletnik.si/4_4/000/000/19f/152/La-14TM.pdf. Accessed online on January 21, 2013).

1.4.2. Lactobacillus bulgaricus

Lactobacillus delbrueckii subsp. (subspecies) bulgaricus (since 1984 known as Lactobacillus bulgaricus). It is one of several bacteria used in the production of yoghurt and other fermented products.

This is a long, threaded Gram-positive stick. It is immobile, does not form spores and grows at low pH (about 5,4 - 4,6). L. delbrueckii spp. bulgaricus is commonly used in the production of yoghurt in combination with Streptococcus thermophilus (Courtin et. al., 2003). Two Lactobaclillus species work synergistically. Both species produce lactic acid, which gives the yoghurt a sour taste and acts as a preservative (Zourari et al.,1992). L. bulgaricus is a homofermentative lactic acid bacterium that ferments glucose, lactose, fructose and sometimes galactose or mannose. Growth temperature up to 48-50 ⁰C. (Kandler, Weiss, 1986). L. bulgaricus probiotic activity is associated with their ability to produce antimicrobial substances such as bacteriocins, organic acids and hydrogen peroxide (Stamatova, 2010).

1.4.3. Lactobacillus rhamnosus

A well-known probiotic bacterium Lactobacillus GG protects and restores food antigens (Isolauri et al. 1993; Malin et al. 1997) or drugs (Gotteland et al. 2001) increased mucosal permeability. This bacterium stimulates intestinal mucosal secretion (Mack et al. 2003) and protects epithelial cell tight junctions from damage in the presence of E. coli infection (Khaled et al. 2003).

Lactic acid bacterium Lactobacillus GG (L. rhamnosus ATCC 53103) is the best-known and best-studied probiotic. It remains viable in the gastrointestinal tract environment, colonises the intestinal mucosa and epithelial cells, and strengthens the immune defence against harmful bacteria and viruses. When taken regularly Lactobacillus GG adapts to the normal bacterial flora. It increases not only lactic acid but also bifidobacteria. Lactobacillus GG has a positive effect on the metabolic processes of the gastrointestinal tract, inhibiting the formation of harmful substances in the large intestine and binding toxins. Inhibits the growth and adhesion of potentially harmful bacteria.

Lactobacillus rhamnosus  the ability to survive in the human digestive tract (Doron  et. al., 2005).

1.4.4. Lactobacillus plantarum

Lactobacillus plantarum  - non-pathogenic, 2-4 μm long, Gram-positive, aerobic rods that naturally occur in the human gastrointestinal tract. Small, tending to form chains, often with square ends, mesophyll. Optimum development temperature 30 ^oC - 35 ^oC, minimum - 10 ^oC - 15 ^oC, maximum - 45 ^oC - 50 ^oC. Temperature-resistant: 60 ^oC, kills within 10 minutes, but can withstand short-term heating 72 ^oC. It ferments sucrose and maltose and produces lactic acid from lactose. L. plantarum resistant to the effects of bile and NaCl, can develop at 6 % of the normal range. NaCl (Masteikienė, 2002; Bixquert 2009). They are found in various fermented foods such as milk, meat, fish, plants; fermented feeds; and in the digestive tract of humans and animals.

 L. plantarum is also found in many types of cheese (Vanderhoof, 2001). Vanderhoof emphasised L. plantarum the ability to create the right conditions to prevent enterocolitis in rats, and to effectively treat children with a lack of good bacteria in their gut. It has been shown that in addition to improving digestion L. plantarum is effective in the treatment of irritable bowel syndrome, colitis and Crohn's disease (Vriesa et. al., 2006). Niedzielin et. al. found that people with irritable bowel syndrome treated with L. plantarum, a significant reduction in abdominal pain (Niedzelin et. al., 1998). L. plantarum has the ability to destroy pathogens and preserve important nutrients, vitamins and antioxidants, and has the ability to produce L-lysine, a beneficial amino acid. Latest from L. plantarum Studies have shown that these bacteria can be effective in preventing soy allergies. Researchers at the University of Illinois conducted two studies in 2008. They fermented soya seeds, flour and food using micro-organisms. Fermented and unfermented soy products were introduced into the blood plasma of people allergic to soy. L. plantarum caused the greatest reduction in the immune response to soy products. E. Hauschildt, in studies with laboratory animals, found that this probiotic improved liver function, reduced the risk of stomach inflammation, reduced bloating in patients with irritable bowel syndrome, increased carboxylic acid levels in faeces, and reduced fibrinogen levels in the blood (Hauschildt 2001). L. plantarum the colony lives in the gut, prevents pathogens from attaching to the mucosal wall, competes for food, and removes harmful bacteria from the body without causing damage. These bacteria are also resistant to many antibiotics (Vriesa et. al., 2006).

Lactobacillus plantarum produces the following bacteriocins: plantaricin A (PlnA), PlnE, PlnF, PlnJ, PlnKit PlnN (Anderssen et. al.., 1998).  L. plantarum produces plantaricin C, which kills mast cells by acting through the cytoplasmic membrane, is bactericidal in sensitive cultures and in some cases causes cell lysis. Bacteriocins often have a narrow inhibitory spectrum, but lactobacilli compensate for this by producing several bacteriocins that belong to different classes and have different inhibitory spectra (Klaenhammer, 1988; Eijsink et. al., 2002).

Some Lactobacilluss strains produce antibacterial substances, bacteriocins, with different activities for different Yersinia enterocolitica, Salmonella, E. coli O157:H7 and E. coli K88 strains. These strains were significantly more susceptible L. plantarum synthesised bacteriocins. L. plantarum also produce antifungal substances (Laniewska - Moroz et. al.., 2001).

It also has the unique property of attaching to the intestinal mucosa via mannose receptors, which is not characteristic of Gram+ batteries but is characteristic of Gram-. This property enables L. plantarum compete with pathogenic bacteria (e.g. Salmonella) (Adlerberth et. al., 1996). Adhesion of pathogenic bacteria is the most important mechanism by which they become established in the gut (Patti, Höök, 1994).

Nissen et.al. found in vitro that L. plantarum may improve intestinal integrity, the metabolic activity of intestinal cells and stimulate the immune response (Nissen et al. 2009).

Evidence shows that the effect of antibiotics on the digestive tract is reduced by L. plantarum consumption (Lonnermark et al. 2009). It also found that L. plantarum may protect intestinal epithelial cells damaged by E. coli (Qin et al. 2009).

L. plantarum Widely used in the food industry and as a probiotic in medicine (http://www.ipm-

int.org/boxmode/pdf/lactobacillus_plantarum.pdf. Accessed on 02.10.2012).

1.4.5. Lactobacillus fermentum

Lactobacillus fermentum are mesophilic, medium-sized rod-shaped bacteria 0.5-0.7 μm thick to 5 μm long, either singly or in chains. Their optimum development temperature is 18-30 ^oC, minimum - 10 ⁰C, maximum - 45 ⁰C. They ferment various sugars such as glucose, lactose, raffinose, maltose, and produce up to 1.2% lactic acid, acetic acid, ethyl alcohol and carbon dioxide. Widespread on plants, used for fermentation of fruits, vegetables, ensiling of feed (Masteikienė, 2002; Songisepp  et al., 2005 ).  

L. fermentum is an obligate heterofermentative lactic acid bacterium (Canchaya et al.., 2003) Most strains produce bacteriocins and break down the hard-to-digest sugars raffinose and stachyose (Songisepp et al.., 2005).

It survives well in the digestive and intestinal tracts of animals and humans. Produces lysozyme, a gas from glucose and NH₃ from arginine. Catalase-negative, non-spore-forming, immobile, fermenting ribose, galactose, D-glucose, D-tagatose and gluconate (http://enologyaccess.org. Accessed 19.12.2013).

Lactobacillus fermentum Increases production of secretory anti-Salmonella IgA, activates phagocytes and reduces Salmonella colonisation of the liver and other organs (Reid et. al., 2002). 

In vitro experiments have shown that L. fermentum inactivates up to 10¹⁰ adenoviruses and vesicular stomatitis virions within 10 min, with an effect similar to that of acid treatment (Candieux et. al., 2002).

Of the human microflora, only lactobacilli L. fermentum can produce lysozyme, a member of a family of factors that have a significant role in protecting human mechanisms against infectious agents. After all, secretory antibodies bind complement and become bacteriocidal only in the presence of lysozyme, and lysozyme is involved in the regulation of the microflora (Jankauskiene 1996). L. fermentum Lysozyme production improves the adhesive properties of lactobacilli, enhances immunomodulatory, antianemic, regeneration-stimulating and other biological properties (Shenderov, 1996)

It found that one L. fermentum strains are able to attach to the gastric mucosal epithelium in vitro but not to the epithelium of the small and large intestine, while other strains are able to attach to the epithelium of the small and large intestine. Thus, the adhesive properties of lactobacilli depend on the strain and tissue specificity of the micro-organism (Day and Lisanski, 1990).